Integration of tumor characteristics with breast cancer index

ABSTRACT

Methods of determining risk of recurrence of a breast cancer of a subject are provided. Also provided are methods of predicting responsiveness to a therapy of a breast cancer of a subject. Additionally, methods of recommending treatment for a subject that has breast cancer are provided. Further provided are methods of treating a subject that has breast cancer. Systems for performing described methods are also provided.

CROSS-REFERENCE

This application claims the benefit of U.S. Provisional Application No.62/255,260, filed Nov. 13, 2015, and U.S. Provisional Application No.62/265,964, filed Dec. 10, 2015, all of which are incorporated herein byreference in their entireties.

BACKGROUND OF THE INVENTION

Estrogen-receptor-positive breast cancer is a disease with a protractedrisk of recurrence. After 5 years of adjuvant tamoxifen, patients have asustained risk of disease recurrence and death for at least 15 yearsafter diagnosis. Long-term follow-up from pivotal upfront trials ofadjuvant aromatase inhibitors, including the Arimidex, Tamoxifen, Aloneor in Combination (ATAC) trial and Breast International Group (BIG) 1-98study (Cuzick et al., 2010), show a continuing rate of recurrence ofabout 2% per year after initial therapy, with greater than half of allrecurrences occurring after 5 years of adjuvant endocrine therapy.

BRIEF SUMMARY OF THE INVENTION

In view of the foregoing, there is a need for extended adjuvant therapyand a biomarker that can guide the treatment decision-making process.The present disclosure generally relates to determination of risk ofrecurrence and response to treatment in breast cancer, includingimproved methods for making those determinations.

The inventors have discovered that a Molecular Grade Index (MGI) andBreast Cancer Index (BCI) can achieve more accurate predictions of riskof recurrence and response to treatments by including tumor size and/ortumor grade characteristics into those determinations. These assays arecalled MGIN+ and BCIN+, respectively.

The present disclosure provides a method of determining risk ofrecurrence of a breast cancer of a subject is provided. In someembodiments, the method comprises:

assaying a sample of breast cancer cells from the subject for theexpression levels of BUB1B, CENPA, NEK2, RACGAP1 and RRM2;

combining the expression levels as a single index into a subject's MGIindex;

determining tumor size and/or grade of the subject's breast cancer;

combining the subject's MGI index with the tumor size and/or grade toobtain the subject's MGIN+ score;

establishing an MGIN+ score cut point below which there is no risk ofrecurrence, determined from MGIN+ indices of a training set of samplesof breast cancer tissue in a representative dataset from patients thathad breast cancer recurrence and did not have breast cancer recurrence;and

comparing the subject's MGIN+ score with the cut point. In someembodiments, the cancer of the subject has a low risk of recurrence ifthe subject's MGIN+ score is below the cut point, and the cancer of thesubject has a high risk of recurrence if the subject's MGIN+ score isabove the cut point.

Also provided is a method of predicting responsiveness to a therapy of abreast cancer of a subject. In some embodiments, the method comprises:

assaying a sample of breast cancer cells from the subject for theexpression levels of BUB1B, CENPA, NEK2, RACGAP1 and RRM2;

combining the expression levels as a single index into a subject's MGIindex;

determining tumor size and/or grade of the subject's breast cancer;

combining the subject's MGI index with the tumor size and/or grade toobtain the subject's MGIN+ score;

establishing an MGIN+ score cut point below which the likelihood ofresponsiveness is below a set level, wherein the MGIN+ score cut pointis determined from MGIN+ indices of a training set of samples of breastcancer tissue in a representative dataset from patients that wereresponsive to the therapy and were not responsive to the therapy;

comparing the subject's MGIN+ score with the cut point. In someembodiments, the cancer of the subject is not likely to be responsive tothe therapy if the subject's MGIN+ score is below the cut point, and thecancer of the subject is likely to be responsive to the therapy if thesubject's MGIN+ score is above the cut point.

Additionally provided is a method of determining risk of recurrence of abreast cancer of a subject. In some embodiments, the method comprises:

assaying a sample of breast cancer cells from the subject for theexpression levels of BUB1B, CENPA, NEK2, RACGAP1 and RRM2;

combining the expression levels as a single index into a subject's MGIindex;

assaying the sample of breast cancer cells from the subject for theexpression levels of HOXB13 and IL17BR;

calculating the ratio of the expression levels of HOXB13/IL17BR toobtain the subject's H/I ratio;

summing the subject's H/I ratio with the subject's MGI index to obtain asubject's BCI index;

determining tumor size and/or grade of the subject's breast cancer;

combining the subject's BCI index with the tumor size and/or grade toobtain the subject's BCIN+ score;

establishing a BCIN+ score cut point below which there is no risk ofrecurrence, wherein the BCIN+ score cut point is determined from BCIN+indices of a training set of samples of breast cancer tissue in arepresentative dataset from patients that had breast cancer recurrenceand did not have breast cancer recurrence; and

comparing the subject's BCIN+ score with the cut point. In someembodiments, the cancer of the subject has a low risk of recurrence ifthe subject's BCIN+ score is below the cut point, and the cancer of thesubject has a high risk of recurrence if the subject's BCIN+ score isabove the cut point.

Further, a method of predicting responsiveness to a therapy of a breastcancer of a subject is provided. In some embodiments, the methodcomprises:

assaying a sample of breast cancer cells from the subject for theexpression levels of BUB1B, CENPA, NEK2, RACGAP1 and RRM2;

combining the expression levels as a single index into a subject's MGIindex;

assaying the sample of breast cancer cells from the subject for theexpression levels of HOXB13 and IL17BR;

calculating the ratio of the expression levels of HOXB13/IL17BR toobtain the subject's H/I ratio;

summing the subject's H/I ratio with the subject's MGI index to obtain asubject's BCI index;

determining tumor size and/or grade of the subject's breast cancer;

combining the subject's BCI index with the tumor size and/or grade toobtain the subject's BCIN+ score;

establishing a BCIN+ score cut point below which the likelihood ofresponsiveness is below a set level, wherein the BCIN+ score cut pointis determined from BCIN+ indices of a training set of samples of breastcancer tissue in a representative dataset from patients that wereresponsive to the therapy and were not responsive to the therapy; and

comparing the subject's BCIN+ score with the cut point. In someembodiments, the cancer of the subject is not likely to be responsive tothe therapy if the subject's BCIN+ score is below the cut point, and thecancer of the subject is likely to be responsive to the therapy if thesubject's BCIN+ score is above the cut point.

The inventors have also discovered that MGI and BCI are effective inpredicting risk of recurrence and responsiveness to treatment insubjects with lymph node positive breast cancer.

Thus, in some embodiments, a method of determining risk of recurrence ofa breast cancer of a subject is provided. In some embodiments, thesubject's breast cancer is lymph node positive. In some embodiments, themethod comprises:

assaying a sample of breast cancer cells from the subject for theexpression levels of BUB1B, CENPA, NEK2, RACGAP1 and RRM2;

combining the expression levels as a single index into a subject's MGIindex;

assaying the sample of breast cancer cells from the subject for theexpression levels of HOXB13 and IL17BR;

calculating the ratio of the expression levels of HOXB13/IL17BR toobtain the subject's H/I ratio;

summing the subject's H/I ratio with the subject's MGI index to obtain asubject's BCI index;

establishing a BCI score cut point below which there is no risk ofrecurrence, determined from BCI indexes of a of a training set ofsamples of breast cancer tissue in a representative dataset frompatients that had breast cancer recurrence and did not have breastcancer recurrence; and

comparing the subject's BCI score with the cut point; wherein the cancerof the subject has a low risk of recurrence if the subject's BCI scoreis below the cut point, and the cancer of the subject has a high risk ofrecurrence if the subject's BCI score is above the cut point.

In some embodiments, the present disclosure provides a method ofpredicting responsiveness to a therapy of a breast cancer of a subject.In some embodiments, the subject's breast cancer is lymph node positive.In some embodiments, the method comprises:

assaying a sample of breast cancer cells from the subject for theexpression levels of BUB1B, CENPA, NEK2, RACGAP1 and RRM2;

combining the expression levels as a single index into a subject's MGIindex;

assaying the sample of breast cancer cells from the subject for theexpression levels of HOXB13 and IL17BR;

calculating the ratio of the expression levels of HOXB13/IL17BR toobtain the subject's H/I ratio;

summing the subject's H/I ratio with the subject's MGI index to obtain asubject's BCI index;

establishing an BCI score cut point below which the likelihood ofresponsiveness is below a set level, determined from BCI indexes of a ofa training set of samples of breast cancer tissue in a representativedataset from patients whose breast cancer was responsive to the therapyand patients whose breast cancer was not responsive to the therapy; and

comparing the subject's BCI score with the cut point. In someembodiments, the cancer of the subject is not likely to be responsive tothe therapy if the subject's BCI score is below the cut point, and thecancer of the subject is likely to be responsive to the therapy if thesubject's BCI score is above the cut point.

In some embodiments, a method of recommending treatment for a subjectthat has breast cancer is provided. In some embodiments, the methodcomprises determining risk of recurrence by one of the above methods,then, if the subject has a low risk of recurrence, recommending notreatment, or if the subject has a high risk of recurrence, (a)recommending treatment with paclitaxel with 5-fluorouracil, doxorubicinand cyclophosphamide if the subject has not previously been treated forbreast cancer, or (b) recommending endocrine therapy if the subject haspreviously been treated for breast cancer.

In some embodiments, a method or treating a subject that has breastcancer that has not been treated is provided. In some embodiments, themethod comprises determining risk of recurrence by one of theabove-described methods, then, if the subject has a low risk ofrecurrence, not treating the subject, or if the subject has a high riskof recurrence, treating the subject with paclitaxel with 5-fluorouracil,doxorubicin and cyclophosphamide.

Additionally provided is a method of recommending treatment for asubject that has breast cancer and has previously had chemotherapy, themethod comprising predicting responsiveness to endocrine therapy by oneof the above-described methods, and recommending treatment of thesubject with endocrine therapy if responsiveness is predicted, orrecommending discontinuation of endocrine therapy if responsiveness isnot predicted.

Also provided is a method of treating a subject that has breast cancer,the method comprising predicting responsiveness to endocrine therapy byone of the above-described methods, and treating the subject withendocrine therapy if responsiveness is predicted, or discontinuingendocrine therapy if responsiveness is not predicted.

Further provided is a method or treating a subject that has breastcancer and has previously had chemotherapy. In some embodiments, themethod comprises determining risk of recurrence by one of theabove-described methods, then, if the subject has a low risk ofrecurrence, the subject is not treated, or if the subject has a highrisk of recurrence, the subject is treated with endocrine therapy.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a graph showing example prognostic information (LR-χ2) for BCIand additional performance of clinical variables (ΔLR-χ2), in accordancewith some embodiments.

FIG. 2 is a histogram of an example BCIN+ with new defined cut-off pointfor low versus high risk, in accordance with some embodiments.

FIG. 3 is a Kaplan-Meier curve and associated HR for example BCIN+ lowversus high risk groups, in accordance with some embodiments.

FIG. 4 provides an example illustration of prognostic value of BCI andimprovement in the prognostic value by the addition of tumor size, tumorgrade, or both, as measured by the change in likelihood ratio statistic,χ² (ΔLR-χ²) in patients with N1 disease, in accordance with someembodiments.

FIGS. 5A-5D illustrates example results for the prognostic performanceof BCIN+ for overall 15-year and late post-5-year distant recurrence inpatients with N1 disease, in accordance with some embodiments. In eachgraph, the top line corresponds to distant recurrence in patientsclassified by BCIN+ as high risk.

FIG. 6 illustrates example results for the rates of overall (0-10 year)and late (5-10 year) distant recurrence as a function of continuousBCIN+, in accordance with some embodiments.

FIG. 7 illustrates example results for univariate and multivariate Coxregression analyses of overall 15-year and post-5-year prognosticperformance of BCIN+, in accordance with some embodiments.

DETAILED DESCRIPTION OF THE INVENTION

As used herein, the singular forms “a”, “an” and “the” are intended toinclude the plural forms as well, unless the context clearly indicatesotherwise. Additionally, the use of “or” is intended to include“and/or”, unless the context clearly indicates otherwise.

A gene expression “pattern” or “profile” or “signature” refers to therelative expression of one or more genes between two or more clinicaloutcomes, cancer outcomes, cancer recurrence outcomes, and/or survivaloutcomes, which is correlated with being able to distinguish betweensaid outcomes. In some cases, the outcome is that of breast cancer.

A “gene” is a polynucleotide that encodes a discrete product, whetherRNA or proteinaceous in nature. It is appreciated that more than onepolynucleotide may be capable of encoding a discrete product. The termincludes alleles and polymorphisms of a gene that encodes the sameproduct, or a functionally associated (including gain, loss, ormodulation of function) analog thereof, based upon chromosomal locationand ability to recombine during normal mitosis.

The terms “correlate” or “correlation” or equivalents thereof refer toan association between expression of one or more genes and a physiologicstate of a cell to the exclusion of one or more other state asidentified by use of the methods as described herein. A gene may beexpressed at a higher or a lower level and still be correlated with oneor more cancer state or outcome.

A “polynucleotide” is a polymeric form of nucleotides of any length,either ribonucleotides or deoxyribonucleotides. This term refers to theprimary structure of the molecule. Thus, this term includes double- andsingle-stranded DNA and RNA. It also includes known types ofmodifications including labels known in the art, methylation, “caps”,substitution of one or more of the naturally occurring nucleotides withan analog, and internucleotide modifications such as uncharged linkages(e.g., phosphorothioates, phosphorodithioates, etc.), as well asunmodified forms of the polynucleotide.

The term “amplify” is used in the broad sense to mean creating anamplification product that can be made enzymatically, such as with DNAor RNA polymerases, for example using polymerase chain reaction (PCR),as is known in the art. “Amplification,” as used herein, generallyrefers to the process of producing multiple copies of a desiredsequence, particularly those of a sample. “Multiple copies” mean atleast 2 copies. A “copy” does not necessarily mean perfect sequencecomplementarity or identity to the template sequence.

By corresponding is meant that a nucleic acid molecule shares asubstantial amount of sequence identity with another nucleic acidmolecule. Substantial amount means at least 90%, 95%, 98%, 99%, or 100%sequence identity, such as can be determined using an alignmentalgorithm, for example the BLAST algorithm, as described in Altschul etal., J. Mol. Biol. 215:403-410 (1990) (using the published defaultsetting, e.g. parameters w=4, t=17). Methods for amplifying mRNA aregenerally known in the art, and include reverse transcription PCR(RT-PCR) and those described in U.S. Pat. No. 6,794,141. Another methodwhich may be used is quantitative PCR (or Q-PCR). Alternatively, RNA maybe directly labeled as the corresponding cDNA by methods known in theart.

A “microarray” is a linear or two-dimensional array of preferablydiscrete regions, each having a defined area, formed on the surface of asolid support such as, but not limited to, glass, plastic, or syntheticmembrane. The density of the discrete regions on a microarray isdetermined by the total numbers of immobilized polynucleotides to bedetected on the surface of a single solid phase support, preferably atleast about 50/cm², more preferably at least about 100/cm², even morepreferably at least about 500/cm², but preferably below about 1,000/cm².Preferably, the arrays contain less than about 500, about 1000, about1500, about 2000, about 2500, or about 3000 immobilized polynucleotidesin total. As used herein, a DNA microarray is an array ofoligonucleotides or polynucleotides placed on a chip or other surfacesused to hybridize to amplified or cloned polynucleotides from a sample.Since the position of each particular group of primers in the array isknown, the identities of a sample polynucleotides can be determinedbased on their binding to a particular position in the microarray.

Some embodiments of the disclosure relate to the identification of genesthat are over- or under-expressed. In some embodiments, methods of thedisclosure involve determining expression by hybridization of mRNA, oran amplified or cloned version thereof, of a sample cell to apolynucleotide that is unique to a particular gene sequence. Preferredpolynucleotides of this type contain at least about 20, at least about22, at least about 24, at least about 26, at least about 28, at leastabout 30, or at least about 32 consecutive nucleotides of a genesequence that is not found in other gene sequences. The term “about” asused in the previous sentence refers to an increase or decrease of 1from the stated numerical value. Even more preferred are polynucleotidesof at least or about 50, at least or about 100, at least about or 150,at least or about 200, at least or about 250, at least or about 300, atleast or about 350, or at least or about 400 nucleotides of a genesequence that is not found in other gene sequences. The term “about” asused in the preceding sentence refers to an increase or decrease of 10%from the stated numerical value. Such polynucleotides may also bereferred to as polynucleotide probes that are capable of hybridizing tosequences of the genes, or unique portions thereof, described herein.Preferably, the sequences are those of mRNA encoded by the genes, thecorresponding cDNA to such mRNAs, and/or amplified versions of suchsequences. In preferred embodiments of the disclosure, thepolynucleotide probes are immobilized on an array, other devices, or inindividual spots that localize the probes.

In some embodiments of the disclosure, all or part of a disclosedsequence may be amplified and detected by methods such as the polymerasechain reaction (PCR) and variations thereof, such as, but not limitedto, quantitative PCR (Q-PCR), reverse transcription PCR (RT-PCR), andreal-time PCR, optionally real-time RT-PCR. Such methods typicallyutilize one or two primers that are complementary to portions of adisclosed sequence, where the primers are used to prime nucleic acidsynthesis. The newly synthesized nucleic acids are optionally labeledand may be detected directly or by hybridization to a polynucleotide ofthe disclosure. The newly synthesized nucleic acids may be contactedwith polynucleotides (containing sequences) of the disclosure underconditions which allow for their hybridization.

In some embodiments of the disclosure, gene expression may be determinedby analysis of expressed protein in a cell sample of interest by use ofone or more antibodies specific for one or more epitopes of individualgene products (proteins) in said cell sample. Such antibodies arepreferably labeled to permit their easy detection after binding to thegene product.

The term “label” refers to a composition capable of producing adetectable signal indicative of the presence of the labeled molecule.Suitable labels include radioisotopes, nucleotide chromophores, enzymes,substrates, fluorescent molecules, chemiluminescent moieties, magneticparticles, bioluminescent moieties, and the like. Non-limiting examplesof labels include compositions detectable by spectroscopic,photochemical, biochemical, immunochemical, electrical, optical orchemical means.

The term “support” refers to conventional supports such as beads,particles, dipsticks, fibers, filters, membranes and silane or silicatesupports such as glass slides.

As used herein, a “cancer tissue sample” or “cancer cell sample” refersto a cell-containing sample of tissue isolated from an individualafflicted with the corresponding cancer. The sample may be from materialremoved via a surgical procedure, such as a biopsy. Such samples areprimary isolates (in contrast to cultured cells) and may be collected byany suitable means recognized in the art. In some embodiments, the“sample” may be collected by a non-invasive method, including, but notlimited to, abrasion or fine needle aspiration.

A “breast tissue sample” or “breast cell sample” refers to a sample ofbreast tissue or fluid isolated from an individual suspected of beingafflicted with, or at risk of developing, breast cancer. Such samplesare primary isolates (in contrast to cultured cells) and may becollected by any non-invasive means, including, but not limited to,ductal lavage, fine needle aspiration, needle biopsy, the devices andmethods described in U.S. Pat. No. 6,328,709, or any other suitablemeans recognized in the art. Alternatively, the “sample” may becollected by an invasive method, including, but not limited to, surgicalbiopsy.

“Expression” and “gene expression” include transcription and/ortranslation of nucleic acid material. The term may also be limited, ifso indicated, as referring only to the transcription of nucleic acids.

As used herein, the term “comprising” and its cognates are used in theirinclusive sense; that is, equivalent to the term “including” and itscorresponding cognates.

Conditions that “allow” an event to occur or conditions that are“suitable” for an event to occur, such as hybridization, strandextension, and the like, or “suitable” conditions are conditions that donot prevent such events from occurring. Thus, these conditions permit,enhance, facilitate, and/or are conducive to the event. Such conditions,known in the art and described herein, depend upon, for example, thenature of the nucleotide sequence, temperature, and buffer conditions.These conditions also depend on what event is desired, such ashybridization, cleavage, strand extension or transcription.

Sequence “mutation,” as used herein, refers to any sequence alterationin the sequence of a gene disclosed herein interest in comparison to areference sequence. A sequence mutation includes single nucleotidechanges, or alterations of more than one nucleotide in a sequence, dueto mechanisms such as substitution, deletion or insertion. Singlenucleotide polymorphism (SNP) is also a sequence mutation as usedherein. Because some embodiments of the present disclosure are based ona relative level of gene expression, mutations in non-coding regions ofgenes as disclosed herein may also be assayed in the practice of thedisclosure.

“Detection” includes any means of detecting, including direct andindirect detection of gene expression and changes therein. For example,“detectably less” products may be observed directly or indirectly, andthe term indicates any reduction (including the absence of detectablesignal). Similarly, “detectably more” product means any increase,whether observed directly or indirectly.

Differences in expression of the disclosed sequences between twoconditions being evaluated (e.g., high or low risk of recurrence) aredefined in the following terms based upon percent or fold changes inexpression between the two conditions. Differences between the twoconditions may be of 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 120, 140,160, 180, or 200%. Fold increases or decreases from one condition to theother condition may be of 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6,6.5, 7, 7.5, 8, 8.5, 9, 9.5, or 10.

A “selective estrogen receptor modulator” or SERM is an “antiestrogen”agent that in some tissues act like estrogens (agonist) but blockestrogen action in other tissues (antagonist). A “selective estrogenreceptor downregulators” (or “SERD”s) or “pure” antiestrogens includesagents which block estrogen activity in all tissues. See e.g. Howell etal. (Best Bractice & Res. Clin. Endocrinol. Metab. 18(1):47-66, 2004).Preferred SERMs of the disclosure are those that are antagonists ofestrogen in breast tissues and cells, including those of breast cancer.Non-limiting examples of such include TAM, raloxifene, GW5638, and ICI182,780. The possible mechanisms of action by various SERMs have beenreviewed (see for example Jordan et al., 2003, Breast Cancer Res.5:281-283; Hall et al., 2001, J. Biol. Chem. 276(40):36869-36872;Dutertre et al. 2000, J. Pharmacol. Exp. Therap. 295(2):431-437; andWijayaratne et al., 1999, Endocrinology 140(12):5828-5840). Othernon-limiting examples of SERMs include triphenylethylenes, such astamoxifen, GW5638, TAT-59, clomiphene, toremifene, droloxifene, andidoxifene; benzothiophenes, such as arzoxiphene (LY353381 orLY353381-HCl); benzopyrans, such as EM-800; naphthalenes, such asCP-336,156; and ERA-923.

Non-limiting examples of SERD or “pure” antiestrogens include agentssuch as ICI 182,780 (fulvestrant or faslodex) or the oral analogueSR16243 and ZK 191703 as well as aromatase inhibitors and chemicalovarian ablation agents as described herein.

Other agents encompassed by SERM as used herein include progesteronereceptor inhibitors and related drugs, such as progestomimetics likemedroxyprogesterone acetate, megace, and RU-486; and peptide basedinhibitors of ER action, such as LH-RH analogs (leuprolide, zoladex,[D-Trp6]LH-RH), somatostatin analogs, and LXXLL motif mimics of ER aswell as tibolone and resveratrol. As noted above, preferred SERMs of thedisclosure are those that are antagonist of estrogen in breast tissuesand cells, including those of breast cancer. Non-limiting examples ofpreferred SERMs include the actual or contemplated metabolites (in vivo)of any SERM, such as, but not limited to, 4-hydroxytamoxifen (metaboliteof tamoxifen), EM652 (or SCH 57068 where EM-800 is a prodrug of EM-652),and GW7604 (metabolite of GW5638). See e.g. Willson et al. (1997,Endocrinology 138(9):3901-3911) and Dauvois et al. (1992, Proc. Nat'l.Acad. Sci., USA 89:4037-4041) for discussions of some specific SERMs.

Other SERMs are those that produce the same relevant gene expressionprofile as tamoxifen or 4-hydroxytamoxifen. One example of means toidentify such SERMs is provided by Levenson et al. (2002, Cancer Res.62:4419-4426).

Unless defined otherwise all technical and scientific terms used hereinhave the same meaning as commonly understood to one of ordinary skill inthe art to which this disclosure belongs.

Risk of recurrence and responsiveness to therapy in breast cancer can bepredicted by measuring expression levels of the genes BUB1B, CENPA,NEK2, RACGAP1, RRM2, HOXB13 and IL17BR; combining the expression levelsof BUB1B, CENPA, NEK2, RACGAP1 and RRM2 to obtain the subject'sMolecular Grade Index (MGI), and combining the subject's MGI with theratio of the expression levels of HOXB13/IL17BR, to obtain the subject'sBreast Cancer Index (BCI). The subject's BCI is then compared to the BCIvalues from patients with known outcomes from a representative dataset.MGI is highly prognostic in patients with estrogen-receptor-positivebreast cancer. HOXB13/IL17BR (H:I ratio) is prognostic for early andlate distant recurrences, and is predictive of extended adjuvantaromatase inhibitor benefit in patients with early-stageestrogen-receptor-positive breast cancer. See for example U.S. Pat. Nos.7,930,105 and 7,504,214; US Patent Publications 2011/0183858,2011/0136680, 2013/0281502, and 2015/0203921; and PCT Patent PublicationWO/2012/079059. In some cases, integration of clinicopathologicalfactors with other molecular scores can show better prognosticperformance than the molecular scores alone (see e.g. Dowsett et al.,2010, J. Clin. Oncol. 28:1829-1834; and Filipits et al., 2011, ClinCancer Res. 17:6012-6020).

The inventors have discovered that, by combining MGI or BCI with tumorclinical data such as tumor size and/or grade, a more accurateprediction of risk of recurrence and responsiveness is possible.Exemplary procedures for combining MGI or BCI with tumor clinical data,to determine an MGIN+ score or BCIN+ score, respectively, are providedherein, such as in the exmaples.

In some embodiments, a method of determining risk of recurrence of abreast cancer of a subject is provided. In some embodiments, the methodcomprises:

assaying a sample of breast cancer cells from the subject for theexpression levels of BUB1B, CENPA, NEK2, RACGAP1 and RRM2;

combining the expression levels as a single index into a subject's MGIindex;

determining tumor size and/or grade of the subject's breast cancer;

combining the subject's MGI index with the tumor size and/or grade toobtain the subject's MGIN+ score;

establishing an MGIN+ score cut point below which there is no risk ofrecurrence, determined from MGIN+ indices of a training set of samplesof breast cancer tissue in a representative dataset from patients thathad breast cancer recurrence and did not have breast cancer recurrence;and

comparing the subject's MGIN+ score with the cut point. In someembodiments, the cancer of the subject has a low risk of recurrence ifthe subject's MGIN+ score is below the cut point, and the cancer of thesubject has a high risk of recurrence if the subject's MGIN+ score isabove the cut point.

Also provided is a method of predicting responsiveness to a therapy of abreast cancer of a subject. In some embodiments, the method comprises:

assaying a sample of breast cancer cells from the subject for theexpression levels of BUB1B, CENPA, NEK2, RACGAP1 and RRM2;

combining the expression levels as a single index into a subject's MGIindex;

determining tumor size and/or grade of the subject's breast cancer;

combining the subject's MGI index with the tumor size and/or grade toobtain the subject's MGIN+ score;

establishing an MGIN+ score cut point below which the likelihood ofresponsiveness is below a set level, wherein the MGIN+ score cut pointis determined from MGIN+ indices of a training set of samples of breastcancer tissue in a representative dataset from patients that wereresponsive to the therapy and were not responsive to the therapy;

comparing the subject's MGIN+ score with the cut point. In someembodiments, the cancer of the subject is not likely to be responsive tothe therapy if the subject's MGIN+ score is below the cut point, and thecancer of the subject is likely to be responsive to the therapy if thesubject's MGIN+ score is above the cut point.

Additionally provided is a method of determining risk of recurrence of abreast cancer of a subject. In some embodiments, the method comprises:

assaying a sample of breast cancer cells from the subject for theexpression levels of BUB1B, CENPA, NEK2, RACGAP1 and RRM2;

combining the expression levels as a single index into a subject's MGIindex;

assaying the sample of breast cancer cells from the subject for theexpression levels of HOXB13 and IL17BR;

calculating the ratio of the expression levels of HOXB13/IL17BR toobtain the subject's H/I ratio;

summing the subject's H/I ratio with the subject's MGI index to obtain asubject's BCI index;

determining tumor size and/or grade of the subject's breast cancer;

combining the subject's BCI index with the tumor size and/or grade toobtain the subject's BCIN+ score;

establishing a BCIN+ score cut point below which there is no risk ofrecurrence, wherein the BCIN+ score cut point is determined from BCIN+indices of a training set of samples of breast cancer tissue in arepresentative dataset from patients that had breast cancer recurrenceand did not have breast cancer recurrence; and

comparing the subject's BCIN+ score with the cut point. In someembodiments, the cancer of the subject has a low risk of recurrence ifthe subject's BCIN+ score is below the cut point, and the cancer of thesubject has a high risk of recurrence if the subject's BCIN+ score isabove the cut point.

Further, a method of predicting responsiveness to a therapy of a breastcancer of a subject is provided. The method comprises

assaying a sample of breast cancer cells from the subject for theexpression levels of BUB1B, CENPA, NEK2, RACGAP1 and RRM2;

combining the expression levels as a single index into a subject's MGIindex;

assaying the sample of breast cancer cells from the subject for theexpression levels of HOXB13 and IL17BR;

calculating the ratio of the expression levels of HOXB13/IL17BR toobtain the subject's H/I ratio;

summing the subject's H/I ratio with the subject's MGI index to obtain asubject's BCI index;

determining tumor size and/or grade of the subject's breast cancer;

combining the subject's BCI index with the tumor size and/or grade toobtain the subject's BCIN+ score;

establishing a BCIN+ score cut point below which the likelihood ofresponsiveness is below a set level, wherein the BCIN+ score cut pointis determined from BCIN+ indices of a training set of samples of breastcancer tissue in a representative dataset from patients that wereresponsive to the therapy and were not responsive to the therapy; and

comparing the subject's BCIN+ score with the cut point. In someembodiments, the cancer of the subject is not likely to be responsive tothe therapy if the subject's BCIN+ score is below the cut point, and thecancer of the subject is likely to be responsive to the therapy if thesubject's BCIN+ score is above the cut point.

In some of these embodiments, both the tumor size and grade are combinedand contribute to the MGIN+ or BCIN+. See e.g. Example 1.

These methods can be utilized with any breast cancer, including estrogenreceptor positive (ER+) and estrogen receptor negative (ER−) breastcancer. Additionally, these methods are useful for lymph node negative(LN−) or lymph node positive (LN+) breast cancer. In some embodiments, 1to 3 lymph nodes are positive (see e.g. Example 1).

Some embodiments of these methods are also useful for a subject that hasbeen treated for breast cancer, e.g., with endocrine or chemotherapy,for any length of time. In some embodiments, the subject has had 5 yearsof endocrine therapy.

In some embodiments where the subject has been treated withchemotherapy, the chemotherapy comprised treatment with paclitaxel. Insome of these embodiments, the chemotherapy also or alternativelycomprised treatment with 5-fluorouracil, doxorubicin andcyclophosphamide.

Methods where response to a therapy is predicted are not limited topredicting response to any particular therapy, including anychemotherapy, any endocrine therapy, radiation therapy and surgery. Insome embodiments, response to endocrine therapy, for example totamoxifen or anastrozole, is predicted. In some embodiments, theendocrine therapy is with a selective estrogen receptor modulator(SERM), e.g., tamoxifen. In other embodiments, the endocrine therapy iswith a selective estrogen receptor down-regulator (SERD). In someembodiments, the endocrine therapy is with an aromatase inhibitor (AI),e.g., anastrozole, exemestane or letrozole.

In some embodiments, responsiveness to chemotherapy is determined. Insome embodiments, responsiveness to treatment with paclitaxel ispredicted. Optionally, such chemotherapy further comprises treatmentwith 5-fluorouracil, doxorubicin and cyclophosphamide.

In some embodiments, responsiveness to radiation therapy is predicted.

Methods for predicting likelihood of recurrence can encompass anyrecurrence, e.g., distant recurrence or local recurrence.

In some embodiments, the BCIN+ is calculated by assessing the individualresponsiveness or risk of cancer recurrence as part of a continuousBCIN+ variable, wherein the individual responsiveness or risk ofrecurrence increases in a linear relationship with the BCIN+ variable.See e.g. US Patent Publication 2015/0203921.

In some embodiments of the various methods described herein, anyappropriate analytical model can be used to combine the subject's MGIgene expression levels as a single index into a subject's index usescoefficients determined from Cox proportional hazards regression, anaccelerated failure time model, a parametric survival model, a logisticmodel, or a linear discriminant analysis. Non-limiting examples of aparametric survival model includes an exponential survival model and aWeibull survival model. In some embodiments, the coefficients aredetermined from Cox proportional hazard models.

In some embodiments where the method predicts likelihood of recurrence,the method comprises establishing a cut point for low versus high riskgroups that ensures the low risk patients will have minimal 10-yearresidual disease. This can be established by setting the cut point belowwhere any samples in the training set had recurrence. In thoseembodiments, a larger training set would provide for greater likelihoodthat no recurrence will occur.

The inventors have also discovered that MGI and BCI are effective inpredicting risk of recurrence and responsiveness to treatment in lymphnode positive subjects.

In some embodiments, a method of determining risk of recurrence of abreast cancer of a subject is provided. In some embodiments, thesubject's breast cancer is lymph node positive. In some embodiments, themethods comprise:

assaying a sample of breast cancer cells from the subject for theexpression levels of BUB1B, CENPA, NEK2, RACGAP1 and RRM2;

combining the expression levels as a single index into a subject's MGIindex;

assaying the sample of breast cancer cells from the subject for theexpression levels of HOXB13 and IL17BR;

calculating the ratio of the expression levels of HOXB13/IL17BR toobtain the subject's H/I ratio;

summing the subject's H/I ratio with the subject's MGI index to obtain asubject's BCI index;

establishing a BCI score cut point below which there is no risk ofrecurrence, determined from BCI indexes of a of a training set ofsamples of breast cancer tissue in a representative dataset frompatients that had breast cancer recurrence and did not have breastcancer recurrence; and

comparing the subject's BCI score with the cut point; wherein the cancerof the subject has a low risk of recurrence if the subject's BCI scoreis below the cut point, and the cancer of the subject has a high risk ofrecurrence if the subject's BCI score is above the cut point.

In some embodiments, a method of predicting responsiveness to a therapyof a breast cancer of a subject. In some embodiments, the subject'sbreast cancer is lymph node positive. In some embodiments, the methodcomprises:

assaying a sample of breast cancer cells from the subject for theexpression levels of BUB1B, CENPA, NEK2, RACGAP1 and RRM2;

combining the expression levels as a single index into a subject's MGIindex;

assaying the sample of breast cancer cells from the subject for theexpression levels of HOXB13 and IL17BR;

calculating the ratio of the expression levels of HOXB13/IL17BR toobtain the subject's H/I ratio;

summing the subject's H/I ratio with the subject's MGI index to obtain asubject's BCI index;

establishing a BCI score cut point below which the likelihood ofresponsiveness is below a set level, determined from BCI indexes of a ofa training set of samples of breast cancer tissue in a representativedataset from patients whose breast cancer was responsive to the therapyand patients whose breast cancer was not responsive to the therapy; and

comparing the subject's BCI score with the cut point. In someembodiments, the cancer of the subject is not likely to be responsive tothe therapy if the subject's BCI score is below the cut point, and thecancer of the subject is likely to be responsive to the therapy if thesubject's BCI score is above the cut point.

The above discussion of the methods comprising MGIN+ or BCIN+ alsoapplies to these methods.

Methods described herein may be used to aid in the selection oftreatment, such as among endocrine therapy, chemotherapy, radiationtherapy, surgery or any combination thereof. In some embodiments, thedisclosure includes compositions and methods for determining theexpression levels of the MGIN+ or BCIN+ genes and the tumor data, or BCIwith LN+ subjects, as a predictor of endocrine therapy effectiveness. Insome cases, the predictor may be of responsiveness or non-responsivenessto a SERM, such as tamoxifen, or an SERD. This includes cases whereassay of a breast cancer cell containing sample from a subject reveals alow MGIN+ or BCIN+, indicating the likelihood of non-responsiveness totamoxifen. In other cases, the predictor may be of the effectiveness ofone form of endocrine therapy over another. This includes determiningMGIN+ or BCIN+ as an indicator of greater responsiveness to an aromataseinhibitor (AI) in comparison to tamoxifen or another SERM or an SERD.Non-limiting examples of an AI include non-steroidal inhibitors such asletrozole and anastrozole and irreversible steroidal inhibitors such asexemestane.

In some embodiments, the disclosure provides compositions and methodsfor the use MGIN+, BCIN+ or BCI as a predictor of chemotherapy treatmentoutcome. The MGIN+, BCIN+ or BCI determination may be used to predictchemo-sensitivity, such as to paclitaxel/FAC (paclitaxel followed by5-fluorouracil, doxorubicin and cyclophosphamide) or taxol oranthracyclin therapy as non-limiting examples. In some embodiments, thedisclosure provides determining MGIN+ or BCIN+, where a high MGIN+ orBCIN+ value is an indicator of increased likelihood of a completepathological response (pCR) to chemotherapy, such as post-operative(post-surgical intervention) treatment with paclitaxel/FAC as anon-limiting example. As a non-limiting example, the detecting may be ofexpression in a cancer cell from a pre-operative cell containing sampleused to diagnose cancer in the subject.

In some embodiments, a method of recommending treatment for a subjectthat has breast cancer is provided. In some embodiments, the methodcomprises determining risk of recurrence by one of the above methods,then, if the subject has a low risk of recurrence, recommending notreatment, or if the subject has a high risk of recurrence, (a)recommending treatment with paclitaxel with 5-fluorouracil, doxorubicinand cyclophosphamide if the subject has not previously been treated forbreast cancer, or (b) recommending endocrine therapy if the subject haspreviously been treated for breast cancer. In some of these embodiments,the subject has previously had chemotherapy and endocrine therapy isrecommended. These embodiments may further comprise treating the subjectwith the recommended treatment.

In some embodiments, a method or treating a subject that has breastcancer that has not been treated is provided. In some embodiments, themethod comprises determining risk of recurrence by one of theabove-described methods, then, if the subject has a low risk ofrecurrence, not treating the subject, or if the subject has a high riskof recurrence, treating the subject with paclitaxel with 5-fluorouracil,doxorubicin and cyclophosphamide.

Additionally provided is a method of recommending treatment for asubject that has breast cancer and has previously had chemotherapy, themethod comprising predicting responsiveness to endocrine therapy by oneof the above-described methods, and recommending treatment of thesubject with endocrine therapy if responsiveness is predicted, orrecommending discontinuation of endocrine therapy if responsiveness isnot predicted. Optionally, where responsiveness is not predicted, thesubject is treated with (a) surgery and chemotherapy and/or (b)radiation is recommended.

Also provided is a method of treating a subject that has breast cancer,the method comprising predicting responsiveness to endocrine therapy byone of the above-described methods, and treating the subject withendocrine therapy if responsiveness is predicted, or discontinuingendocrine therapy if responsiveness is not predicted. Optionally, whereresponsiveness is not predicted, the subject is treated with (a) surgeryand chemotherapy and/or (b) radiation is recommended.

Further provided is a method or treating a subject that has breastcancer and has previously had chemotherapy. In some embodiments, themethod comprises determining risk of recurrence by one of the abovemethods, then, if the subject has a low risk of recurrence, the subjectis not treated, or if the subject has a high risk of recurrence, thesubject is treated with endocrine therapy.

To determine the expression levels of genes, any method known in the artmay be utilized. In some embodiments, expression based on detection ofmRNA which hybridizes to the genes identified and disclosed herein isused. This is readily performed by any mRNA detection oramplification+detection method known or recognized as equivalent in theart such as, but not limited to, reverse transcription PCR, the methodsdisclosed in U.S. Pat. No. 6,794,141, and methods to detect thepresence, or absence, of RNA stabilizing or destabilizing sequences. Insome embodiments, the mRNA is converted into cDNA.

In some embodiments, the ability to discriminate is conferred by theidentification of expression of the individual genes as relevant and notby the form of the assay used to determine the actual level ofexpression. An assay may utilize any identifying feature of anidentified individual gene as disclosed herein as long as the assayreflects, quantitatively or qualitatively, expression of the gene in the“transcriptome” (the transcribed fraction of genes in a genome) or the“proteome” (the translated fraction of expressed genes in a genome).Identifying features include, but are not limited to, unique nucleicacid sequences used to encode (DNA), or express (RNA), said gene orepitopes specific to, or activities of, a protein encoded by said gene.In some embodiments, it is sufficient to provide the identity of thegene(s) used to discriminate between cancer outcomes and an appropriatecell containing sample for use in an expression assay. Similarly, thenature of the cell containing sample is not limiting, as fresh tissue,freshly frozen tissue, and fixed tissue, such as formalin-fixedparaffin-embedded (FFPE) tissues, may be used in the disclosed methods.

Expression based on detection of a presence, increase, or decrease inprotein levels or activity may also be used. Detection may be performedby any immunohistochemistry (IHC) based, blood based (especially forsecreted proteins), antibody (including autoantibodies against theprotein) based, exfoliate cell (from the cancer) based, massspectroscopy based, and image (including used of labeled ligand) basedmethod known in the art and recognized as appropriate for the detectionof the protein. Antibody and image based methods are additionally usefulfor the localization of tumors after determination of cancer by use ofcells obtained by a non-invasive procedure (such as ductal lavage orfine needle aspiration), where the source of the cancerous cells is notknown. A labeled antibody or ligand may be used to localize thecarcinoma(s) within a patient.

Some embodiments use a nucleic acid based assay to determine expressionby immobilization of one or more sequences of the genes identifiedherein on a solid support, including, but not limited to, a solidsubstrate such as an array or to beads or bead-based technology.Alternatively, solution based expression assays known in the art mayalso be used.

The immobilized gene(s) may be in the form of polynucleotides that areunique or otherwise specific to the gene(s) such that the polynucleotidewould be capable of hybridizing to a DNA or RNA corresponding to thegene(s). These polynucleotides may be the full length of the gene(s) orbe short sequences of the genes (up to one nucleotide shorter than thefull length sequence known in the art, e.g., by deletion from the 5′ or3′ end of the sequence) that are optionally minimally interrupted (suchas by mismatches or inserted non-complementary basepairs) such thathybridization with a DNA or RNA corresponding to the gene(s) is notaffected. In some cases, the polynucleotides used are from the 3′ end ofthe gene, such as within about 350, about 300, about 250, about 200,about 150, about 100, or about 50 nucleotides from the polyadenylationsignal or polyadenylation site of a gene or expressed sequence.

The skilled person is fully capable of aligning any two or more of theknown expressed sequences for each of these genes to identify an area ofidentity or conserved changes as a region that uniquely identifies eachof these genes in comparison to other genes. Furthermore, the skilledperson is fully capable of aligning any two or more of the knownexpressed sequences for each of these genes to identify an area uniqueto one or more of the of the expressed sequences as a region thatuniquely identifies one known expressed sequence relative to at leastone other expressed sequence. As a non-limiting example, a unique regionmay be in a variant of the expressed sequence for one of the known genessuch that the region may be used to identify expression of the variant.

The sequences of the same genes have also been identified andcharacterized from other animal species. Thus the skilled person in thefield is clearly aware of how to identify the disclosed genes relativeto other animal genes. The skilled person may also optionally comparethe known sequences of the disclosed genes from different animal sourcesto identify conserved regions and sequences unique to these genesrelative to other genes.

Polynucleotides containing mutations relative to the sequences of thedisclosed genes may also be used so long as the presence of themutations still allows hybridization to produce a detectable signal. Theimmobilized gene(s) may be used to determine the state of nucleic acidsamples prepared from sample cancer, or breast, cell(s) for which theoutcome of the sample's subject (e.g. patient from whom the sample isobtained) is not known or for confirmation of an outcome that is alreadyassigned to the sample's subject. Without limiting the disclosure, sucha cell may be from a patient with ER+ breast cancer. The immobilizedpolynucleotide(s) need only be sufficient to specifically hybridize tothe corresponding nucleic acid molecules derived from the sample undersuitable conditions.

As will be appreciated by those skilled in the art, some of thecorresponding sequences noted above include 3′ polyA (or polyT on thecomplementary strand) stretches that do not contribute to the uniquenessof the disclosed sequences. The disclosure may thus be practiced withsequences lacking the 3′ polyA (or polyT) stretches. The uniqueness ofthe disclosed sequences refers to the portions or entireties of thesequences which are found only in the disclosed gene's nucleic acids,including unique sequences found at the 3′ untranslated portion of thegenes. Preferred unique sequences for the practice of the disclosure arethose which contribute to the consensus sequences for each of the threesets such that the unique sequences will be useful in detectingexpression in a variety of individuals rather than being specific for apolymorphism present in some individuals. Alternatively, sequencesunique to an individual or a subpopulation may be used. The preferredunique sequences are preferably of the lengths of polynucleotides of thedisclosure as discussed herein.

Methods to identify increased RNA stability (resulting in an observationof increased expression) or decreased RNA stability (resulting in anobservation of decreased expression) may also be used. These methodsinclude the detection of sequences that increase or decrease thestability of mRNAs containing the genes' sequences.

These methods also include the detection of increased mRNA degradation.In some embodiments of the disclosure, polynucleotides having sequencespresent in the 3′ untranslated and/or non-coding regions of the abovedisclosed sequences are used to detect expression levels of the genesequences in cancer, or breast, cells. Such polynucleotides mayoptionally contain sequences found in the 3′ portions of the codingregions of the above disclosed sequences.

Polynucleotides containing a combination of sequences from the codingand 3′ non-coding regions preferably have the sequences arrangedcontiguously, with no intervening heterologous sequences.

In some embodiments, methods of the disclosure may be practiced withpolynucleotides having sequences present in the 5′ untranslated and/ornon-coding regions of the gene sequences in cancer, or breast, cells todetect their levels of expression. Such polynucleotides may optionallycontain sequences found in the 5′ portions of the coding regions.Polynucleotides containing a combination of sequences from the codingand 5′ non-coding regions preferably have the sequences arrangedcontiguously, with no intervening heterologous sequences. The disclosuremay also be practiced with sequences present in the coding regions ofthe disclosed gene sequences.

Non-limiting polynucleotides contain sequences from 3′ or 5′untranslated and/or non-coding regions of at least about 20, at leastabout 22, at least about 24, at least about 26, at least about 28, atleast about 30, at least about 32, at least about 34, at least about 36,at least about 38, at least about 40, at least about 42, at least about44, or at least about 46 consecutive nucleotides. The term “about” asused in the previous sentence refers to an increase or decrease of 1from the stated numerical value. Even more preferred are polynucleotidescontaining sequences of at least or about 50, at least or about 100, atleast about or 150, at least or about 200, at least or about 250, atleast or about 300, at least or about 350, or at least or about 400consecutive nucleotides. The term “about” as used in the precedingsentence refers to an increase or decrease of 10% from the statednumerical value.

In some embodiments, sequences from the 3′ or 5′ end of the abovedescribed coding regions as found in polynucleotides of the disclosureare of the same lengths as those described above, except that they wouldnaturally be limited by the length of the coding region. The 3′ end of acoding region may include sequences up to the 3′ half of the codingregion. Conversely, the 5′ end of a coding region may include sequencesup to the 5′ half of the coding region. Of course the above describedsequences, or the coding regions and polynucleotides containing portionsthereof, may be used in their entireties.

Polynucleotides combining the sequences from a 3′ untranslated and/ornon-coding region and the associated 3′ end of the coding region may beat least or about 100, at least about or 150, at least or about 200, atleast or about 250, at least or about 300, at least or about 350, or atleast or about 400 consecutive nucleotides. Preferably, thepolynucleotides used are from the 3′ end of the gene, such as withinabout 350, about 300, about 250, about 200, about 150, about 100, orabout 50 nucleotides from the polyadenylation signal or polyadenylationsite of a gene or expressed sequence. Polynucleotides containingmutations relative to the sequences of the disclosed genes may also beused so long as the presence of the mutations still allows hybridizationto produce a detectable signal.

In some embodiments of the disclosure, polynucleotides containingdeletions of nucleotides from the 5′ and/or 3′ end of the abovedisclosed sequences may be used. The deletions are preferably of 1-5,5-10, 10-15, 15-20, 20-25, 25-30, 30-35, 35-40, 40-45, 45-50, 50-60,60-70, 70-80, 80-90, 90-100, 100-125, 125-150, 150-175, or 175-200nucleotides from the 5′ and/or 3′ end, although the extent of thedeletions would naturally be limited by the length of the sequences andthe need to be able to use the polynucleotides for the detection ofexpression levels.

Some polynucleotides of the disclosure from the 3′ end of the abovedisclosed sequences include those of primers and optional probes forquantitative PCR. In some embodiments, the primers and probes are thosewhich amplify a region less than about 350, less than about 300, lessthan about 250, less than about 200, less than about 150, less thanabout 100, or less than about 50 nucleotides from the from thepolyadenylation signal or polyadenylation site of a gene or expressedsequence.

In some embodiments, polynucleotides containing portions of the abovedisclosed sequences including the 3′ end are used. Such polynucleotidescan contain at least or about 50, at least or about 100, at least aboutor 150, at least or about 200, at least or about 250, at least or about300, at least or about 350, or at least or about 400 consecutivenucleotides from the 3′ end of the disclosed sequences.

The disclosure also includes polynucleotides used to detect geneexpression in breast cancer cells. The polynucleotides may comprise ashorter polynucleotide consisting of sequences found in the above genesin combination with heterologous sequences not naturally found incombination with the sequences. Non-limiting examples include shortsequences from cloning vectors or present in restriction fragments usedto prepare labeled probes or primers as described herein.

The requisite level of expression may be that which is identified by themethods described herein for the genes used. Additionally, the assayingmay include preparing RNA from the sample, optionally for use in PCR(polymerase chain reaction) or other analytical methodology as describedherein. The PCR methodology is optionally RT-PCR (reversetranscription-PCR) or quantitative PCR, such as real-time RT-PCR.Alternatively, the assaying may be conducted by use of an array, such asa microarray, by next-generation sequencing, or by any other methodknown in the art. Optionally, the sample of cancer cells is dissectedfrom tissue removed or obtained from said subject. As described herein,a variety of sample types may be used, including a formalin fixedparaffin embedded (FFPE) sample as a non-limiting example.

The term “cut point” as used herein indicates the cut-off value abovewhich MGIN+, BCIN+ or BCI is considered a high value and below whichthose indices are considered a low value.

By way of non-limiting example, the genes for MGIN+, BCIN+ or BCI may beassayed and used to detect expression levels that, when factoring intumor clinical data, correspond to a value that is “high risk” (which isabove the cut point) for MGIN+, BCIN+ or BCI, or to detect expressionlevels that correspond to a value that is “low risk” (which is at orbelow the cut point) for MGIN+, BCIN+ or BCI, as disclosed herein. Insome cases, the MGIN+, BCIN+ or BCI cut point threshold may be 1. Inalternative embodiments, the cutoff may be at or about 2, at or about 3,at or about 4, at or about 5, at or about 6, at or about 7, at or about8, at or about 9, at or about 10, greater than 10, or any value inbetween. In some embodiments, the cutoff is greater than 1, 2, 3, 4, 5,6, 7, 8, or 9. With respect to the H:I ratio, its determination may bemade as described in Ma et al., Cancer Cell. 2004 June; 5(6):607-16.Also see e.g. Ma et al., Clin Cancer Res 2008; 14(9): 2601-8; and Ma etal., J Clin Oncol 2006; 24(28): 4611-9.

The cut point value can be determined without undue experimentation byusing known methods, for example by using training sets, e.g. asdescribed in Example 1. The cut point can be selected to achieve thesensitivity (SE), specificity (SP), positive predictive value (PPV) andnegative predictive value (NPV) desired.

For the evaluation of methods of determining risk of recurrence orresponse to therapy, categorizations can be used to interpret theresults of tests in the clinical setting. The diagnostic or prognosticvalue of a method can be defined by its SE, SP, PPV and NPV. Any testmethod will produce True Positives (TP), False Negatives (FN), FalsePositives (FP), and True Negatives (TN). The “sensitivity” of a test isthe percentage of all patients with disease present or that do respondwho have a positive test or (TP/TP+FN)×100%. The “specificity” of a testis the percentage of all patients without disease or who do not respond,who have a negative test or (TN/FP+TN)×100%. The “predictive value” or“PV” of a test is a measure (%) of the times that the value (positive ornegative) is the true value, i.e., the percent of all positive teststhat are true positives is the Positive Predictive Value (PPV) or(TP/TP+FP)×100%. The “negative predictive value” (NPV) is the percentageof patients with a negative test who will not respond or(TN/FN+TN)×100%. Another measure, the “accuracy” or “efficiency” of atest, is the percentage of the times that the test gives the correctanswer compared to the total number of tests or(TP+TN/TP+TN+FP+FN)×100%. The “error rate” calculates from thosepatients predicted to respond who did not and those patients whoresponded that were not predicted to respond or(FP+FN/TP+TN+FP+FN)×100%. The overall test “specificity” is a measure ofthe accuracy of the sensitivity and specificity of a test do not changeas the overall likelihood of disease changes in a population, thepredictive value does change. The PV changes with a physician's clinicalassessment of the presence or absence of disease or presence or absenceof clinical response in a given patient.

For any given test, the TP, FN, FP and TN can be determined and adjustedby the skilled artisan without undue experimentation by, e.g., adjustingthe cut point value, adjusting the statistical significance (e.g., the Pvalue) for making the clinical determination; adjusting the accuracy ofthe test procedures, etc.

Using a cut point of 7 as a non-limiting example for MGIN+, BCIN+, orBCI, the disclosed methods provide two possible assay outcomes for agiven sample: high risk or responsive MGIN+, BCIN+, or BCI,corresponding to a value above 7, and low risk or nonresponsive MGIN+,BCIN+, or BCI, corresponding to a value below 7. A high risk orresponsive MGIN+, BCIN+, or BCI is indicative of a high risk ofrecurrence of breast cancer, or responsive to a particular therapy,depending on the clinical parameter under examination. A low MGIN+,BCIN+, or BCI is indicative of a low risk of recurrence, ornonresponsive to the therapy under consideration.

In some embodiments, an MGIN+, BCIN+, or BCI intermediate-risk of cancerrecurrence may be included.

As a non-limiting example, the cancer cell may be one from apre-operative histological sample, or biopsy, used to diagnose cancer inthe subject. For such a subject with ductal carcinoma in situ (DCIS),the current standard of care is surgery, with breast conserving surgerypreferred over a radical mastectomy, to remove the DCIS. This is oftenfollowed by post-operative radiotherapy, optionally with endocrinetherapy, such as treatment with tamoxifen, a selective estrogen receptormodulator (SERM), a selective estrogen receptor down-regulator (SERD),an aromatase inhibitor (AI) such as letrozole, a targeted therapy suchas anti-mTOR therapy (e.g., with Afinitor®) or anti-HER2 therapy (e.g.,with Herceptin®) and/or chemotherapy, using any compound known in theart.

In some cases, the disclosed methods may be used to select or eliminatetherapies for premenopausal women, or for postmenopausal women,diagnosed with cancer. Premenopausal women include those who are lessthan about 35 years of age. In these subjects, high MGIN+, BCIN+, or BCIis an indicator of cancer recurrence or response to therapy. The methodmay include assaying a breast cancer cell containing sample from asubject for expression of these genes. As a non-limiting example, thecell may be one from a pre-operative histological sample used todiagnose cancer in the subject.

The methods may include identifying the premenopausal subject as likely,or unlikely, to experience cancer recurrence, and optionally furtherinclude adjusting treatment modalities for the subject to address theexpected outcome. As a non-limiting example, determination of a lowlikelihood of recurrence may be used to confirm the suitability of, orto select, breast conserving therapies, optionally with reduction inpost-operative therapies like radiation and/or endocrine therapy orchemotherapy. As another non-limiting example, determination of a highlikelihood of recurrence may be used to confirm the suitability of, orto select, radical treatment modalities with inclusion of post-operativetherapies, such as radiation and/or endocrine therapy or chemotherapy.

The determination of MGIN+, BCIN+, or BCI may be in any suitable cellcontaining sample as described herein. Non-limiting examples of cellsfor use in the disclosure include those freshly isolated from thesubject, those frozen after isolation, and those that are fixed and/orembedded, such as formalin fixed, paraffin embedded (FFPE). In mostembodiments, the cells are breast cells, such as breast cancer cells.

In some embodiments, MGIN+, BCIN+, or BCI is used to determine risk ofcancer recurrence in a breast cancer afflicted patient. Non-limitingexamples of late recurrence include after 5 years of treatment with anaromatase inhibitor, targeted therapy or endocrine therapy, such astamoxifen, but also includes after 4 years, after 3 years, or after 2years or less time of treatment. Similarly, the MGIN+ or BCIN+ may beused to predict responsiveness to an anti-aromatase therapy, such asanastrozole or letrozole, targeted therapy or anti-estrogen therapyafter the above time periods.

In some embodiments, the methods disclosed herein can be advantageouslyused on a breast cancer cell-containing sample from a subject, such as aDCIS sample, although the methods described herein can be used with anytype of breast cancer, including any non-invasive, or invasive breastcancer, such as invasive ductal carcinoma, invasive lobular carcinoma,inflammatory breast cancer, male breast cancer, metastatic breastcancer, recurrent breast cancer, papillary carcinoma, triple-negativebreast cancer, Paget's disease of the nipple, sarcoma of the breast,medullary carcinoma, tubular carcinoma, mucinous carcinoma, metaplasticcarcinoma, adenocystic carcinoma, phyllodes tumor and angiosarcoma.

In some embodiments, the present disclosure provides systems forperforming any of the methods described herein. Systems can comprise oneor more modules, each configured to perform one or more steps of amethod disclosed herein. In some embodiments, the system comprises oneor more computer processors. In some embodiments, the system furthercomprises a reaction module in communication with the computerprocessor, wherein the reaction module performs a reaction for measuringgene expression. Processors can be associated with one or morecontrollers, calculation units, and/or other units of a computer system,or implanted in firmware as desired. If implemented in software, theroutines can be stored in any computer readable memory such as in RAM,ROM, flash memory, a magnetic disk, a laser disk, or other storagemedium. Software can be delivered to a computing device via any knowndelivery method including, for example, over a communication channelsuch as a telephone line, the internet, a wireless connection, etc., orvia a transportable medium, such as a computer readable disk, flashdrive, etc. The various steps can be implemented as various blocks,operations, tools, modules or techniques which, in turn, can beimplemented in hardware, firmware, software, or any combination thereof.When implemented in hardware, some or all of the blocks, operations,techniques, etc. can be implemented in, for example, a custom integratedcircuit (IC), an application specific integrated circuit (ASIC), a fieldprogrammable logic array (FPGA), a programmable logic array (PLA), etc.In some embodiments, the computer is configured to receive a userrequest to measure expression levels of one or more genes in a sample.The computer may receive the user request directly (e.g. by way of aninput device such as a keyboard, mouse, or touch screen operated by auser entering a user request) or indirectly (e.g. through a wired orwireless connection, including over the internet). Non-limiting examplesof users include the subject providing the sample, medical personnel,clinicians, laboratory personnel, insurance company personnel, or othersin the health care industry.

In some embodiments, the system generates a report of results obtainedin accordance with a method of the present disclosure. The report can betransmitted to a recipient, such as a user. The report can becommunicated to a recipient at a local or remote location using anysuitable communication medium. For example, the communication medium canbe a display, a network connection, a wireless connection, or aninternet connection. A report can be transmitted over such networks orconnections (or any other suitable means for transmitting information,including but not limited to mailing a printed report) for receipt by arecipient. The recipient can be but is not limited to a subject, ahealth care provider, a health care manager, or electronic system (e.g.one or more computers, and/or one or more servers). In some embodiments,the system sends the report to a recipient's device, such as a personalcomputer, phone, tablet, or other device.

Example embodiments are described in the following Examples. Otherembodiments within the scope of the claims herein will be apparent toone skilled in the art from consideration of the specification orpractice of the invention as disclosed herein. It is intended that thespecification, together with the examples, be considered exemplary only,with the scope and spirit of the invention being indicated by theclaims, which follow the examples.

EXAMPLE 1 Integration of Tumor Size and Grade with the Breast CancerIndex for the Prediction of Distant Recurrence in HR+ Breast Cancer with1-3 Positive Lymph Nodes

This example describes a gene expression-based assay that combines twoindependent biomarkers: the HOXB13:IL17BR expression ratio (H/I), whichinterrogates estrogen signaling, and the Molecular Grade Index (MGI),which quantifies tumor proliferation (see e.g. Zhang et al., 2013, ClinCancer Res 19, 4196-4205). The combination is referred to as a BreastCancer Index (BCI). MGI utilizes the combined expression of five genes,BUB1B, CENPA, NEK2, RACGAP1 and RRM2. BCI significantly predicted 10year distant recurrence (DR) beyond the clinicopathological factors inestrogen receptor positive (ER+), lymph node negative (LN−) breastcancer patients in three cohorts (see e.g. Zhang et al., 2013; Sgroi etal., 2013, Lancet Oncol. 14:1067-1076). BCI also was prognostic in lymphnode positive (LN+) patients in transATAC and MA.14 (Sgroi et al.,2013). Here, a distinct BCI model that integrates tumor size and gradewas evaluated for prediction of DR in women with 1-3 lymph node positivedisease.

219 primary tumor samples from hormonal receptor-positive patients with1-3 positive lymph nodes treated with 5 years of tamoxifen oranastrozole were examined. Women with four or more positive lymph nodeswere excluded. BCI was combined with tumor size and grade into acomprehensive risk score, BCIN+. Kaplan-Meier (KM) estimates of 10 yearDR and hazard ratios (HR) and 95% confidence intervals (CI) wereestimated. Change in likelihood ratio χ² (LR−Δχ²) values were used tomeasure prognostic information of each variable alone or combined in anew score. New cutpoints for low versus high risk groups for the newmodel were determined to ensure the low risk patients had minimal10-year residual disease.

In 219 LN+ patients, BCI alone provided substantial additionalprognostic information to tumor size (LR−Δχ²=11.83, P=0.0006) and grade(LR−Δχ²=8.33, P=0.004). Both clinical variables provided additionalsignificant prognostic information to BCI alone (BCI alone: LR−Δχ²=9.59,P=0.0004; T: LR−Δχ²=7.09, P=0.008; G: LR−Δχ²=27.59, P<0.0001).Integration of tumor size and grade with BCI (BCIN+) provided additionalhighly significant prognostic information compared to BCI alone(Interquartile HR=3.15 [95% CI: 1.54-6.04]; LR−Δχ²=33.89, P<0.0001). Acut-point for a very low risk group in this LN+ population wasdetermined, and included 55 (25%) women with no DR within 10 years. Incontrast, 51 (31.1%) women developed a DR in the high risk group(N=164). 10-year DR risk for those in the high risk group was 35.4% (95%CI 28.0-44.1%).

Integration of tumor size and grade significantly enhanced theprognostic ability of BCI to predict 10 year DR risk in hormonalreceptor-positive patients with 1-3 positive nodes. A significant numberof patients have been identified to have a very low 10-year risk for DR,who may choose to safely forego unnecessary adjuvant chemotherapy orextended adjuvant endocrine therapy.

This examples describes development of a distinct BCI model thatintegrates tumor size and grade for 1-3 LN+ postmenopausal women.

Formalin-fixed, paraffin-embedded (FFPE) blocks of primary tumor werecollected from HR+ patients in the transATAC cohort for RNA extraction(extraction by Genomic Health Inc.).

Primary analysis: HR-positive, LN+ (1-3 nodes only), no chemotherapy, 5years of tamoxifen or anastrozole alone (N=219).

Primary endpoint was distant recurrence (DR).

Statistical analyses included Cox proportional hazard models,Kaplan-Meier analyses, and Likelihood Ratio (LR) tests.

The following models were fitted for those with assessed grade (N=209):

BCI alone

BCI+ tumor size

BCI+ grade

BCI+ tumor size + grade

New cut-off points were defined for low vs. high risk groups.

TABLE 1 Baseline characteristics for all, well, and moderate/poorlydifferentiated LN+ (1-3) LN+ (1-3) well moderate/poorly LN+ (1-3)differentiated differentiated (N = 209) (N = 42) (N = 167) Age (years),median 66.9 (60.0-73.4) 62.3 (57.6-69.7) 68.1 (61.1-73.5) (IQR) BMI(kg/m²), median 26.2 (23.6-29.2) 24.6 (21.9-28.0) 26.7 (24.1-29.8) (IQR)Tumor size (cm),  2.1 (1.7-2.8)  2.1 (1.7-2.8)  2.2 (1.8-3.0) median(IQR) Grade* Well   42 (20.1%)   42 (100%) — Moderate  124 (56.6%)    124 (74.3%) Poor   43 (19.6%)      43 (25.7%) Radiotherapy  157 (71.7%)  29 (69.1%)  123 (73.7%) Mastectomy  121 (55.3%)   20 (47.6%)   92(55.1%) Distant recurrence   51 (23.3%) 0   51 (30.5%) *For 10 tumorsgrade was not assessable

Age and BMI were significantly different (P=0.03, P=0.01, resp.) betweenwomen with well and moderate/poorly differentiated tumors.

All DR were observed in women with 1-3 LN+ and moderate or poorlydifferentiated tumors (Table 1).

BCI was strongly prognostic in women with 1-3 LN+ disease (LR−χ²=8.63,P=0.003) (FIG. 1).

Both the addition of tumor size alone (ΔLR−χ²=7.29, P=0.007) andspecifically grade alone (ΔLR−χ²=23.96, P<0.001) to the BCI improved theprognostic performance significantly.

The addition of both clinicopathological variables to the BCI performedstatistically the best compared to BCI alone (ΔLR−χ²=30.25, P<0.001)(FIG. 1).

Tumor size and grade were integrated into the BCI by use of multivariateCox-proportional model.

A cut-off point for low versus high risk groups was defined (low risk:BCIN+<6.93) that classified around 20% of patients into the low riskgroup (FIG. 2).

No DR was observed for women categorized into the low risk BCIN+ group.All 51 DR (30.5%) were observed in the high risk BCIN+ group (HR forcontinuous BCIN+=2.76 (1.55-4.92)) (FIG. 3). The 10-year DR risk forthose in the high risk group was 35% (95% CI 27.6%-43.6%).

This BCIN+ model, integrating tumor size and grade, statisticallyenhanced the prognostic performance of BCI in postmenopausal women with1-3 LN+ disease.

A significant proportion of women with 1-3 LN+ disease were categorizedto have very low risk 10-year DR risk by BCIN+. These women might bespared unnecessary chemotherapy or extended adjuvant endocrine therapy.

Women categorized as BCIN+ high risk had a significantly higher risk ofDR (35%) during 10 years of follow-up. These women clearly identified ashigh risk by BCIN+ may benefit from additional therapy.

EXAMPLE 2 Validation of BCIN+ Model for Predicting Distant Recurrence inHormone Receptor-Positive (HR+) Breast Cancer with 1-3 Positive Nodes

Women diagnosed with HR+, invasive breast cancer with 1-3 positive nodes(N1 disease) between 1993 and 2007 who received adjuvant endocrinetherapy (ET) with or without chemotherapy, at least 5 years offollow-up, and available FFPE tumor blocks were selected for study(N=402). Median follow-up was 12 years. Average patient age was 53years. Patients included both pre- and post-menopausal women (39%<50years old; 61%≧50 years old). 81% of patients were treated with adjuvantchemotherapy. All patients were treated with adjuvant ET (TAM=48%;AI=17%; TAM+AI sequence=35%), with 276 treated with up to 5 years ofadjuvant ET. Most patients received adjuvant chemotherapy (81%). Mostpatients had ductal histology (86%). The majority of tumors were T1(62%) or T2+ (35%). 17%, 56%, and 26% of tumors were grade 1, 2, and 3,respectively. Additional baseline characteristics of this studypopulation are provided in Table 2.

TABLE 2 Characteristic Category N (%) Age at Surgery <50 yr 156 (39%)≧50 yr 246 (61%) Tumor Size ≦20 mm 249 (62%) >20 mm 153 (38%) TumorGrade Well  70 (17%) Moderate 226 (56%) Poor 106 (26%) ER StatusNegative  4 (1%) Positive 398 (99%) PR Status Negative  37 (9%) Positive364 (91%) Unknown  1 (0%) HER2 Status Negative 259 (64%) Positive  52(13%) Unknown  91 (23%) Surgery Mastectomy 162 (40%) Lumpectomy 240(60%) Tumor Type Ductal 347 (86%) Lobular  45 (11%) Ductal + Lobular  10(3%) Adjuvant Chemotherapy No  76 (19%) Yes 324 (81%) Unknown  2 (0%)Adjuvant ET Tamoxifen (TAM) 191 (48%) Aromatase Inhibitor (AI)  69 (17%)TAM + AI sequence 142 (35%) Duration of Adjuvant Initial Adjuvant Only(<5 yr) 276 (69%) Endocrine Therapy Extended (≧5 yr) 126 (31%) DistantRecurrence Early (<5 yr)  49 (56%) Late (≧5 yr)  38 (44%) All RecurrenceEarly (<5 yr)  51 (55%) Late (≧5 yr)  41 (45%)

Gene expression analyses of formalin-fixed paraffin-embedded (FFPE)samples were performed, while blinded to clinical outcome. For eachcase, three 10 μm tissue sections were cut, and an H&E slide was used toconfirm 40% content of invasive cancer before manual macrodissection toenrich tumor content for RNA extraction. Total RNA was reversetranscribed, the resulting cDNA pre-amplified using the PreAmp MasterMix Kit per manufacturer's instructions (Applied Biosystems, Carlsbad,Calif., USA), followed by TaqMan RT-PCR as described by Ma et al., ClinCancer Res 2008; 14(9): 2601-8. MGI, H/I, and BCI were calculated asdescribed in Ma et al. 2008 and Ma et al., J Clin Oncol 2006; 24(28):4611-9, blinded to all other variables. The primary endpoint was time todistant recurrence, defined as the time from diagnosis to the time offirst metastasis at distant organs. Death before distant recurrence wasconsidered a censoring event. Locoregional recurrences were notconsidered either as events or as censoring events. Late distantrecurrences were defined as distant recurrences occurring after 5 yearsfrom diagnosis and were evaluated within the subset of patients who hadremained distant recurrence free for at least 5 years.

BCIN+ risk scores and categories (“low” and “high”) were determined asin Example 1, including a pre-specified cutpoint, and analyses wereblinded to clinical outcomes. Distant recurrence-free survival for thetwo pre-specified risk groups was evaluated by Kaplan-Meier survivalanalysis, including estimates of overall (0-15 years) and late (≧5years) distant recurrence (DR). The equality of the survival curves wasevaluated with log-rank test. Multivariate Cox proportional hazardmodels were used to evaluate whether BCIN+, as a continuous risk index,provided prognostic information. The multivariate analyses were adjustedfor age, tumor type, surgery type, PR status, and HER2 status. Patientswith missing data on the covariates were excluded from the multivariateanalyses. Inter-quartile Hazard ratios (HR) for continuous BCIN+correspoinding to a change from the 25th to 75th percentile of thedistribution, and corresponding 95% confidence intervals (CI) were alsocalculated. A 2-sided p-value of less than 0.05 was consideredstatistically significant. All analyses were performed using Stata(version 13.1) or R statistical package (version 3.1.3,www.r-project.org).

In multivariate analysis, BCIN+ was significantly associated with riskof overall DR (HR 13.1; 95% CI 3.6-43.9; p<0.0001) and late DR (HR 5.0;95% CI 1.5-16.5; p=0.009). FIG. 4 illustrates prognostic value of BCIand improvement in the prognostic value by the addition of tumor size,tumor grade, or both, as measured by the change in likelihood ratiostatistic, χ² (ΔLR−χ²) in patients with N1 disease. The initial valuefor BCI alone was 20.12 (shaded portion of the bars). The addition oftumor size (ΔLR−χ²=13.29; P=0.0003), grade (ΔLR−χ²=12.72; P=0.004), orboth tumor size and grade (ΔLR−χ²=22.28; P=0.0001), in combination withBCI significantly improved the prognostic performance in patients withN1 disease (unshaded portion of the bars).

Results for the prognostic performance of BCIN+ for overall 15-year andlat post-5-year distant recurrence in patients with N1 disease areillustrated in FIGS. 5A-5D. For risk of overall cumulative distantrecurrence, BCIN+ classified 20% of patients as low risk (N=81), with a1.3% risk of distant recurrence at both the 10-year and 15-yearfollow-up (15-year 95% CI 0.0-3.7%); whereas patients classified asBCIN+ high risk had 24.2% risk of distant recurrence at the 10-yearfollow-up, and a 29% risk of distant recurrence at the 15-year follow-up(15-year 95% CI 23.2-34.4%), giving an HR of 25.93 (95% CI 3.61-186.22)(FIG. 5A). In patients that were distant recurrence-free at year 5 (N=349), BCIN+ classified 23% as low risk, with a 1.3% risk of latedistant recurrence at both the 10-year and 15-year follow-up (95% CI0.0-3.7%); whereas patients classified as BCIN+ high risk had a 10.5%risk of late distant recurrence at the 10-year follow-up, and a 16.1%risk at the 15-year follow-up (95% CI 10.6-21.3%), giving an HR of 12.39(95% CI 1.7-90.35) (FIG. 5B). Similar results were observed in theHER2-subset for overall 15-year risk of distant recurrence and risk oflate (5-15 year) distant recurrence (FIGS. 5C and 5D, respectively).These results indicate that a classification of low risk according to aBCIN+ model carries such a low risk of late recurrence (1.3%) thatcontinued endocrine therapy beyond year 5 is unlikely to substantiallyreduce that risk of recurrence.

FIG. 6 illustrates results for the rates of overall (0-10 year) and late(5-10 year) distant recurrence as a function of continuous BCIN+. Theplots show that risk of distant recurrence increased monotonically asBCIN+ increased for both overall and late distant recurrence. Resultsfor univariate and multivariate Cox regression analyses of overall15-year and post-5-year prognostic performance of BCIN+ are provided inFIG. 7. Further with regard to FIG. 7, age was by 10-year increments,tumor type was ductal vs. non-ductal; surgery was mastectomy vs.lumpectomy; PR was positive vs. negative; chemotherapy was yes vs. no;BCIN+ was a continuous variable per inter-quartile range. Patients withmissing data on the covariates were excluded from the multivariateanalyses. As a continuous variable, BCIN+ was a highly significantprognostic factor with an HR or 2.14 (95% CI 1.56-2.94, P<0.0001) and1.66 (95% CI 1.15-2.38, P =0.006) for overall and late distantrecurrence, respectively.

BCIN+ can provide additional prognostic information to facilitateselection of N+ patients for extended endocrine treatment. In accordancewith some embodiments, patients identified as BCIN+ low may beconsidered adequately treated with adjuvant therapy alone.

EXAMPLE 3 Subset Analysis of Impact of Treatment History on BCIN+Prognostic Ability

The patient population of Example 2 was analyzed to assess the impact oftreatment history on the prognostic ability of a BCIN+ model as inExample 1. BCIN+ risk scores were determined and patients stratifiedinto low risk or high risk categories using a pre-specified cut-point,blinded to clinical outcome. Kaplan-Meier survival analysis was used toestimate overall (0-15 year) and late (5-15 year) distant recurrence(DR), and the difference was evaluated by log-rank test.Treatment-specific subsets were analyzed based on adjuvant endocrine(tamoxifen (TAM)) only versus any history of aromatase inhibitors (AI),and adjuvant chemotherapy treatment history. As explained in Example 2,BCIN+ classified 20% of patients in this population as low risk, and 80%as high risk.

In patients treated with TAM only, BCIN+ low risk and high risk hadsignificantly different 15-year rates of DR of 4.0% (95% CI 0.0-11.4%)versus 41.7% (95% CI 33.0-49.3%), respectively (p=0.0005). For patientsdisease-free at year 5, rates of late DR (5-15 y) were 4.0% (95% CI0.0-11.5%) versus 20.0% (95% CI 11.4-27.8%), respectively (p=0.04). Inpatients treated with an AI, BCIN+ low risk and high risk hadsignificantly different 15-year rates of DR of 0% (95% CI 0.0-0.0%)versus 15.0% (95% CI 8.1-21.5%), respectively (p=0.006). For patientsdisease-free at year 5, rates of late DR were 0.0% (95% CI 0.0-0.0%)versus 12.2% (95% CI 5.6-18.3%), respectively (p=0.02). There was nostatistically significant difference in the prognostic performance ofBCIN+ between patients treated with TAM only versus those with treatmentincluding any history of AI (interaction p=0.99).

In the subset of patients treated with chemotherapy, BCIN+ classified19% and 81% of patients as low and high risk with significantlydifferent 15-year rates of DR of 1.7% (95% CI 0.0-4.9%) versus 30.9%(95% CI 24.4-36.8%), respectively (p<0.0001). For patients disease-freeat year 5, rates of late DR were 1.7% (95% CI 0.0-4.9%) and 16.3% (95%CI 10.2-21.9%), respectively (p=0.006).

These results demonstrate an ability of BCIN+ to identify a subset ofpatients with significantly low risk of recurrence across adjuvantendocrine and chemotherapy treatment backgrounds. BCIN+ can provideadditional prognostic information to facilitate selection of N+ patientsfor extended endocrine treatment. In accordance with some embodiments,patients identified as BCIN+ low may be considered adequately treatedwith adjuvant therapy alone.

In view of the above, it will be seen that several objectives of theinvention are achieved and other advantages attained.

As various changes could be made in the above methods and compositionswithout departing from the scope of the invention, it is intended thatall matter contained in the above description and shown in theaccompanying drawings shall be interpreted as illustrative and not in alimiting sense.

All references cited in this specification are hereby incorporated byreference. The discussion of the references herein is intended merely tosummarize the assertions made by the authors and no admission is madethat any reference constitutes prior art. Applicants reserve the rightto challenge the accuracy and pertinence of the cited references.

What is claimed is:
 1. A method of determining risk of recurrence of abreast cancer of a subject, the method comprising assaying a sample ofbreast cancer cells from the subject for the expression levels of BUB1B,CENPA, NEK2, RACGAP1 and RRM2; combining the expression levels as asingle index into a subject's MGI index; determining tumor size and/orgrade of the subject's breast cancer; combining the subject's MGI indexwith the tumor size and/or grade to obtain the subject's MGIN+ score;establishing an MGIN+ score cut point below which there is no risk ofrecurrence, determined from MGIN+ indices of a training set of samplesof breast cancer tissue in a representative dataset from patients thathad breast cancer recurrence and did not have breast cancer recurrence;and comparing the subject's MGIN+ score with the cut point; wherein thecancer of the subject has a low risk of recurrence if the subject'sMGIN+ score is below the cut point, and the cancer of the subject has ahigh risk of recurrence if the subject's MGIN+ score is above the cutpoint.
 2. A method of predicting responsiveness to a therapy of a breastcancer of a subject, the method comprising assaying a sample of breastcancer cells from the subject for the expression levels of BUB1B, CENPA,NEK2, RACGAP1 and RRM2; combining the expression levels as a singleindex into a subject's MGI index; determining tumor size and/or grade ofthe subject's breast cancer; combining the subject's MGI index with thetumor size and/or grade to obtain the subject's MGIN+ score;establishing an MGIN+ score cut point below which the likelihood ofresponsiveness is below a set level, wherein the MGIN+ score cut pointis determined from MGIN+ indices of a training set of samples of breastcancer tissue in a representative dataset from patients that wereresponsive to the therapy and were not responsive to the therapy;comparing the subject's MGIN+ score with the cut point; wherein thecancer of the subject is not likely to be responsive to the therapy ifthe subject's MGIN+ score is below the cut point, and the cancer of thesubject is likely to be responsive to the therapy if the subject's MGIN+score is above the cut point.
 3. A method of determining risk ofrecurrence of a breast cancer of a subject, the method comprisingassaying a sample of breast cancer cells from the subject for theexpression levels of BUB1B, CENPA, NEK2, RACGAP1 and RRM2; combining theexpression levels as a single index into a subject's MGI index; assayingthe sample of breast cancer cells from the subject for the expressionlevels of HOXB13 and IL17BR; calculating the ratio of the expressionlevels of HOXB13/IL17BR to obtain the subject's H/I ratio; summing thesubject's H/I ratio with the subject's MGI index to obtain a subject'sBCI index; determining tumor size and/or grade of the subject's breastcancer; combining the subject's BCI index with the tumor size and/orgrade to obtain the subject's BCIN+ score; establishing a BCIN+ scorecut point below which there is no risk of recurrence, wherein the BCIN+score cut point is determined from BCIN+ indices of a training set ofsamples of breast cancer tissue in a representative dataset frompatients that had breast cancer recurrence and did not have breastcancer recurrence; and comparing the subject's BCIN+ score with the cutpoint; wherein the cancer of the subject has a low risk of recurrence ifthe subject's BCIN+ score is below the cut point, and the cancer of thesubject has a high risk of recurrence if the subject's BCIN+ score isabove the cut point.
 4. A method of predicting responsiveness to atherapy of a breast cancer of a subject, the method comprising assayinga sample of breast cancer cells from the subject for the expressionlevels of BUB1B, CENPA, NEK2, RACGAP1 and RRM2; combining the expressionlevels as a single index into a subject's MGI index; assaying the sampleof breast cancer cells from the subject for the expression levels ofHOXB13 and IL17BR; calculating the ratio of the expression levels ofHOXB13/IL17BR to obtain the subject's H/I ratio; summing the subject'sH/I ratio with the subject's MGI index to obtain a subject's BCI index;determining tumor size and/or grade of the subject's breast cancer;combining the subject's BCI index with the tumor size and/or grade toobtain the subject's BCIN+ score; establishing a BCIN+ score cut pointbelow which the likelihood of responsiveness is below a set level,wherein the BCIN+ score cut point is determined from BCIN+ indices of atraining set of samples of breast cancer tissue in a representativedataset from patients that were responsive to the therapy and were notresponsive to the therapy; and comparing the subject's BCIN+ score withthe cut point; wherein the cancer of the subject is not likely to beresponsive to the therapy if the subject's BCIN+ score is below the cutpoint, and the cancer of the subject is likely to be responsive to thetherapy if the subject's BCIN+ score is above the cut point.
 5. Themethod of any one of claims 1-4, wherein both the tumor size and gradeare combined.
 6. The method of any one of claims 1-5, wherein thesubject's breast cancer is ER+.
 7. The method of any one of claims 1-6,wherein the subject's breast cancer is lymph node negative.
 8. Themethod of any one of claims 1-6, wherein the subject's breast cancer islymph node positive.
 9. The method of claim 8, wherein 1 to 3 lymphnodes are positive.
 10. The method of any one of claims 1-9, wherein thesubject has been treated for breast cancer.
 11. The method of claim 10,wherein the subject has had 5 years of endocrine therapy.
 12. The methodof claim 10, wherein the subject has had chemotherapy.
 13. The method ofclaim 12, wherein the chemotherapy comprised treatment with paclitaxel.14. The method of claim 12, wherein the chemotherapy further comprisedtreatment with 5-fluorouracil, doxorubicin and cyclophosphamide.
 15. Themethod of claim 2 or 4, wherein the therapy comprises endocrine therapy.16. The method of claim 15, wherein the therapy comprises tamoxifen oranastrozole.
 17. The method of claim 2 or 4, wherein the subject has notbeen treated for breast cancer.
 18. The method of claim 17, whereinpredicting responsiveness to chemotherapy is determined.
 19. The methodof claim 18, wherein the chemotherapy comprises treatment withpaclitaxel.
 20. The method of claim 19, wherein the chemotherapy furthercomprised treatment with 5-fluorouracil, doxorubicin andcyclophosphamide.
 21. The method of claim 2 or 4, wherein predictingresponsiveness to endocrine therapy is determined.
 22. The method ofclaim 21, wherein the endocrine therapy is with a selective estrogenreceptor modulator (SERM).
 23. The method of claim 22, wherein the SERMis tamoxifen.
 24. The method of claim 21, wherein the endocrine therapyis with a selective estrogen receptor down-regulator (SERD).
 25. Themethod of claim 21, wherein the endocrine therapy is with an aromataseinhibitor (AI).
 26. The method of claim 25, wherein the AI isanastrozole, exemestane or letrozole.
 27. The method of 2 or 4, whereinpredicting responsiveness to radiation therapy is determined.
 28. Themethod of claim 1 or 3, wherein the recurrence is distant recurrence.29. The method of any claim 1 or 3, wherein the recurrence is localrecurrence.
 30. The method of any one of claims 3-29, wherein the BCIN+is calculated by assessing the individual responsiveness or risk ofcancer recurrence as part of a continuous BCIN+ variable, wherein theindividual responsiveness or risk of recurrence increases in a linearrelationship with the BCIN+ variable.
 31. The method of any one ofclaims 1-30, wherein combining the subject's expression levels as asingle index into a subject's index uses coefficients determined fromCox proportional hazards regression, an accelerated failure time model,a parametric survival model, a logistic model, or a linear discriminantanalysis.
 32. The method of claim 31, wherein the coefficients aredetermined from Cox proportional hazard models.
 33. The method of claim1 or 3, further comprising establishing a cutpoint for low versus highrisk groups that ensures the low risk patients will have minimal 10-yearresidual disease.
 34. A method of recommending treatment for a subjectthat has breast cancer, the method comprising determining risk ofrecurrence by the method of claim 1 or 3, then if the subject has a lowrisk of recurrence, recommending no treatment, or if the subject has ahigh risk of recurrence, (a) recommending treatment with paclitaxel with5-fluorouracil, doxorubicin and cyclophosphamide if the subject has notpreviously been treated for breast cancer, or (b) recommending endocrinetherapy if the subject has previously been treated for breast cancer.35. The method of claim 34, wherein the subject has previously hadchemotherapy and endocrine therapy is recommended.
 36. The method ofclaim 34 or 35, further comprising treating the subject with therecommended treatment.
 37. A method or treating a subject that hasbreast cancer that has not been treated, the method comprisingdetermining risk of recurrence by the method of claim 1 or 3, then ifthe subject has a low risk of recurrence, not treating the subject, orif the subject has a high risk of recurrence, treating the subject withpaclitaxel with 5-fluorouracil, doxorubicin and cyclophosphamide.
 38. Amethod of recommending treatment for a subject that has breast cancerand has previously had chemotherapy, the method comprising predictingresponsiveness to endocrine therapy by the method of claim 21, andrecommending treatment of the subject with endocrine therapy ifresponsiveness is predicted, or recommending discontinuation ofendocrine therapy if responsiveness is not predicted.
 39. The method ofclaim 38, wherein treatment of the subject with (a) surgery andchemotherapy and/or (b) radiation is recommended if responsiveness isnot predicted.
 40. A method of treating a subject that has breastcancer, the method comprising predicting responsiveness to endocrinetherapy by the method of claim 21, and treating the subject withendocrine therapy if responsiveness is predicted, or discontinuingendocrine therapy if responsiveness is not predicted.
 41. The method ofclaim 40, further comprising treating the subject with (a) surgery andchemotherapy and/or (b) radiation if responsiveness is not predicted.42. A method or treating a subject that has breast cancer and haspreviously had chemotherapy, the method comprising determining risk ofrecurrence by the method of claim 1 or 3, then if the subject has a lowrisk of recurrence, the subject is not treated, or if the subject has ahigh risk of recurrence, the subject is treated with endocrine therapy.43. A method of determining risk of recurrence of a breast cancer of asubject, wherein the subject's breast cancer is lymph node positive, themethod comprising assaying a sample of breast cancer cells from thesubject for the expression levels of BUB1B, CENPA, NEK2, RACGAP1 andRRM2; combining the expression levels as a single index into a subject'sMGI index; assaying the sample of breast cancer cells from the subjectfor the expression levels of HOXB13 and IL17BR; calculating the ratio ofthe expression levels of HOXB13/IL17BR to obtain the subject's H/Iratio; summing the subject's H/I ratio with the subject's MGI index toobtain a subject's BCI index; establishing a BCI score cut point belowwhich there is no risk of recurrence, determined from BCI indexes of aof a training set of samples of breast cancer tissue in a representativedataset from patients that had breast cancer recurrence and did not havebreast cancer recurrence; and comparing the subject's BCI score with thecut point; wherein the cancer of the subject has a low risk ofrecurrence if the subject's BCI score is below the cut point, and thecancer of the subject has a high risk of recurrence if the subject's BCIscore is above the cut point.
 44. A method of predicting responsivenessto a therapy of a breast cancer of a subject, wherein the subject'sbreast cancer is lymph node positive, the method comprising assaying asample of breast cancer cells from the subject for the expression levelsof BUB1B, CENPA, NEK2, RACGAP1 and RRM2; combining the expression levelsas a single index into a subject's MGI index; assaying the sample ofbreast cancer cells from the subject for the expression levels of HOXB13and IL17BR; calculating the ratio of the expression levels ofHOXB13/IL17BR to obtain the subject's H/I ratio; summing the subject'sH/I ratio with the subject's MGI index to obtain a subject's BCI index;establishing an BCI score cut point below which the likelihood ofresponsiveness is below a set level, determined from BCI indexes of a ofa training set of samples of breast cancer tissue in a representativedataset from patients whose breast cancer was responsive to the therapyand patients whose breast cancer was not responsive to the therapy; andcomparing the subject's BCI score with the cut point; wherein the cancerof the subject is not likely to be responsive to the therapy if thesubject's BCI score is below the cut point, and the cancer of thesubject is likely to be responsive to the therapy if the subject's BCIscore is above the cut point.
 45. The method of claim 43 or 44, whereinthe subject's breast cancer is ER+.
 46. The method of any one of claims43-45, wherein 1 to 3 lymph nodes are positive.
 47. The method of anyone of claims 43-46, wherein the subject has been treated for breastcancer.
 48. The method of claim 47, wherein the subject has previouslyhad chemotherapy.
 49. The method of claim 44, wherein the therapycomprises endocrine therapy.
 50. The method of claim 49, wherein thetherapy comprises tamoxifen or anastrozole.
 51. The method of claim 44,wherein the subject has not been treated for breast cancer.
 52. Themethod of claim 51, wherein predicting responsiveness to chemotherapy isdetermined.
 53. The method of claim 52, wherein the chemotherapycomprises paclitaxel.
 54. The method of claim 44, wherein predictingresponsiveness to endocrine therapy is determined.
 55. The method ofclaim 54, wherein the endocrine therapy is with a selective estrogenreceptor modulator (SERM).
 56. The method of claim 55, wherein the SERMis tamoxifen.
 57. The method of claim 54, wherein the endocrine therapyis with a selective estrogen receptor down-regulator (SERD).
 58. Themethod of claim 54, wherein the endocrine therapy is with an aromataseinhibitor (AI).
 59. The method of claim 58, wherein the AI isanastrozole, exemestane or letrozole.
 60. The method of claim 44,wherein predicting responsiveness to radiation therapy is determined.61. The method of claim 43, wherein the recurrence is distantrecurrence.
 62. The method of any claim 43, wherein the recurrence islocal recurrence.
 63. The method of any one of claims 43-62, wherein theBCI is calculated by assessing the individual responsiveness or risk ofcancer recurrence as part of a continuous BCI variable, wherein theindividual responsiveness or risk of recurrence increases in a linearrelationship with the BCI variable.
 64. The method of any one of claims53-53, wherein combining the subject's expression levels as a singleindex into a subject's index uses coefficients determined from Coxproportional hazards regression, an accelerated failure time model, aparametric survival model, an exponential survival model, a Weibullsurvival model, a logistic model, or a linear discriminant analysis. 65.The method of claim 64, wherein the coefficients are determined from Coxproportional hazard models.
 66. The method of claim 43, furthercomprising establishing a cutpoint for low versus high risk groups thatensures the low risk patients had minimal 10-year residual disease. 67.A method of recommending treatment for a subject that has breast cancer,the method comprising determining risk of recurrence by the method ofclaim 43, then if the subject has a low risk of recurrence, recommendingno treatment, or if the subject has a high risk of recurrence, (a)recommending treatment with paclitaxel with 5-fluorouracil, doxorubicinand cyclophosphamide if the subject has not previously been treated forbreast cancer, or (b) recommending endocrine therapy if the subject haspreviously been treated for breast cancer.
 68. The method of claim 67,wherein the subject has had chemotherapy and endocrine therapy isrecommended.
 69. The method of claim 67 or 68, further comprisingtreating the subject with the recommended treatment.
 70. A method ortreating a subject that has breast cancer that has not been treated, themethod comprising determining risk of recurrence by the method of claim43, then if the subject has a low risk of recurrence, not treating thesubject, or if the subject has a high risk of recurrence, treating thesubject with paclitaxel with 5-fluorouracil, doxorubicin andcyclophosphamide.
 71. A method of recommending treatment for a subjectthat has breast cancer and has previously had chemotherapy, the methodcomprising predicting responsiveness to endocrine therapy by the methodof claim 49, and recommending treatment of the subject with endocrinetherapy if responsiveness is predicted, or recommending discontinuationof endocrine therapy if responsiveness is not predicted.
 72. The methodof claim 71, wherein treatment of the subject with (a) surgery andchemotherapy and/or (b) radiation is recommended if responsiveness isnot predicted.
 73. A method of treating a subject that has breastcancer, the method comprising predicting responsiveness to endocrinetherapy by the method of claim 54, and treating the subject withendocrine therapy if responsiveness is predicted, or treating thesubject with (a) surgery and chemotherapy and/or (b) radiation ifresponsiveness is not predicted.
 74. A method or treating a subject thathas breast cancer and has been treated with chemotherapy for 5 years,the method comprising determining risk of recurrence by the method ofclaim 43, then if the subject has a low risk of recurrence, the subjectis not treated, or if the subject has a high risk of recurrence, thesubject is treated with endocrine therapy.